Johnsen L B, Ravn P, Berglund L, Petersen T E, Rasmussen L K, Heegaard C W, Rasmussen J T, Benfeldt C, Fedosov S N
Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Biochemistry. 1998 Sep 8;37(36):12631-9. doi: 10.1021/bi9806697.
Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio. Only the minor form was able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to human tPA as well. The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.
牛组织型纤溶酶原激活剂（tPA）在甲基营养型酵母毕赤酵母中进行了异源表达，并对其结构和动力学进行了表征。在有无纤维蛋白原片段的情况下，研究了牛单链tPA介导的牛纤溶酶原激活作用。我们提出了一种改进的动力学分析新方法，该方法可以研究该过程的稳态和预稳态阶段。研究发现，重组牛tPA存在两种可相互转化的形式，它们以1:50的比例处于平衡状态。只有次要形式能够结合并激活纤溶酶原。为了逆转两种形式之间的平衡，tPA的整个库的饱和需要高浓度的纤溶酶原（Km≥5 microM）。纤维蛋白原片段激活了单链tPA，这是由于该酶的次要“活性”形式优先结合并稳定下来，直到所有tPA分子都被转化。同样的机制也适用于人tPA。在有纤维蛋白原片段存在的情况下，重组牛tPA和人tPA的Km值相似（Km = 0.1 microM），而人tPA的kcat则高5-10倍。
